Chinese people are suffering the highest incidence of hepatocellular carcinoma among the world population. Hepatitis B virus (HBV) causes the acute and chronic hepatitis, and is most likely an important agent to cause hepatocellular carcinoma (Vyas, G. N. et al., Western J. Med. 140:754-762, 1984). HBV envelope contains hepatitis B virus surface antigen (HBsAg) carried with the major polypeptides with a molecular weight 26,000, its glycosylated derivative with a molecular weight of 30,000, and two minor polypeptides with the a molecular weight of 31,000 and 68,000, respectively. The minor polypeptides are carrying the receptor activity for polymerized human serum albumin (pHSA) (Machida, A. et al., Gastroenterology 85:268-274, 1983; Stibbe, W. et al., J. Virol. 46:626-628, 1983; Hansson, B. G. et al., Infect. Immun. 26:125-130, 1979; O'Neill, S. P., J. Med. Virol. 4:177-185. 1979; Towbin, H. et al., Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979).
The purification of HBsAg was reported previously by several other research institutes, and the methods they employed are similar but not identical (Michida, A. et al., supra; Michel, M-L. et al., Biotechnology 3:561-566, 1985; Heermann, K. H. et al., J. of Virol. 52:396-402, 1984). The common principle they used for purification of HBsAg is to apply a series of rate zonal centrifugation, first by sucrose density gradient centrifugation, followed by CsCl density gradient centrifugation. However, by those methods, pre-S containing HBsAgs are purified together with HBsAgs without pre-S peptide.
The present invention develops a simple method for fast and efficiently isolating and purifying pre-S2 containing HBsAgs from the plasma of a single chronic carrier of HBsAg (adw) by ammonium sulfate fractionation, hydroxyapatite column chromatography and pHSA affinity column chromatography. About 500 .mu.g of pre-S2 containing HBsAg was obtained from 140 ml of plasma containing 4,200 .mu.g of HBsAg. Two purified pre-S2 containing HBsAgs were analyzed by SDS-polyacrylamide gel electrophoresis and their molecular weights were determined to be 31,000 and 68,000 respectively.
The present invention revealed that pHSA bound pre-S2 containing HBsAg specifically, and no significant amount of HBsAg (MW 26,000) or its glycosylated derivative (MW 30,000) was adsorbed by pHSA. These results are in an agreement with those of Yu, M. W. et al., (J. Virol. 55:736-743, 1985) or Machida, A. et al supra, and they showed that HBsAg particles were also bound by pHSA, probably due to the presence of pre-S HBsAg in the particles.
Since pHSA binds to pre-S2 containing HBsAg but it does not bind to HBsAg without pre-S, it strongly suggests that pre-S2 region has the binding site for pHSA. Neurath, A. R. et al (Science 224:392:395, 1985) reported that the 55 amino acid residues of pre-S2 region contained the epitopes for immunoglobulin of hepatitis B virus and that the synthetic peptide of the first 26 amino acid terminal residues was shown to induce antibodies. The pre-S2 structure was suggested to be involved in the attachment of HBV to liver cells. Therefore, to induce the antibodies against the infection by HBV, the pre-S2 containing HBsAg could be a much better antigen than HBsAg for preparing the vaccine against hepatitis B virus infection.
The present invention can be applied for the recovery of pre-S2 containing HBsAg produced by recombinant DNA techniques such as the expression of pre-S2 containing HBsAg in Escherichia coli (Fujisawa, Y. et al., Gene 40:23-29, 1985) or Chinese hamster ovary cells (Michel, M-L. et al., supra).
In the present invention, the pHSA-Sepharose 4B affinity column chromatography technique was employed as a major and important step for the purification of pre-S2 containing HBsAg. By using pHSA-Sepharose 4B affinity chromatography, it has the following advantages: (1) it is simple, fast and efficient as described above, (2) it has the specific affinity towards pre-S2 containing HBsAg, and (3) it is superior to immunoadsorbant affinity chromatography, because pHSA is the product made from normal human serum, while for immunoadsorbant affinity technique, the materials are obtained from monoclonal antibody of hybridoma or polyclonal antibody of immuned human being, which are suffered either from the safety or economic problems.